Extract of Acanthopanax koreanum for the treatment or prevention of hepatitis or the liver protective drug

ABSTRACT

The disclosure concerns an extract of  Acanthopanax koreanum  and its use. More particularly, it concerns the extract of  Acanthopanax koreanum  comprising 1) the extract of  Acanthopanax koreanum  extracted from water, 2) among the water extract, the extract of  Acanthopanax koreanum  only containing ethanol insoluble part obtained by precipitating ethanol, 3) among the ethanol insoluble part, the extract of  Acanthopanax koreanum  containing polysaccaride with a molecular weight larger than range of 12,000˜14,000, or 4) among the ethanol insoluble part, the extract of  Acanthopanax koreanum  containing polysaccaride with a molecular weight larger than 100,000, which is respectively obtained from the root or stem of  Acanthopanax koreanum.  The extract of the present invention shows a high inhibitory activity against hepatitis and protects the liver, and thus, can be used for the treatment or prevention of hepatitis, or as a liver protective drug.

FIELD OF THE INVENTION

The present invention relates to an extract of Acanthopanax koreanum forthe treatment or prevention of hepatitis or the liver protective drugand its use.

BACKGROUND OF THE INVENTION

Located between the digestive system and body circulating system inhuman body, liver plays an important role in defending our body from theharmful intrusion of toxic substances and in metabolism. Since foreignsubstances intruded into human body first passes through the liver,liver has a high possibility to be exposed to various toxic substancesother than nutrients, and thus, has a higher possibility to be injuredthan other internal organs.

As an internal organ with excellent restoration ability, liver cancompletely recover its functions in slight damages. However, if liver iscontinuously damaged by alcohol over ingestion, chemical substanceabuse, viral hepatitis, and bile secretion suspension, not only itsfunctions are deteriorated, but a part of liver tissues are completelydamaged, and the thus damaged part cannot be completely restored, whichgoes through liver fibrosis and may finally advance into fatalcirrhosis. Further, liver diseases do not show any pains or subjectivesymptoms at the initial stage, but they are found at the terminal stage.Therefore, it is impossible to treat liver diseases at a proper stage,and thus, liver diseases show a high death rate.

Regardless of the severity of liver diseases, an effective liver-diseasetherapeutic has not yet been found. As for liver diseases caused byviral hepatitis, anti-virus drugs are being used, but their side effectscause serious problems. As for liver diseases caused by toxic substancesrecently increasing due to alcohol and environment pollution, aneffective liver disease therapeutic has not yet been found. Accordingly,the development of a drug, which treats and prevents liver damage whilemaintaining the structure and function of liver tissue is keenlyrequired. However, since no experimental method has been developed tillnow, there are many limitations in developing a liver diseasetherapeutic. That is, in fact, there is not enough experimental supporton the drugs referred to as liver protective drugs.

However, recently, an animal model has been developed which contributedto the development of a liver disease therapeutic. In this connection,an animal model induced with carbon tetrachloride is used in order todevelop a liver disease therapeutic caused by toxic substances, and anacute hepatitis model induced with D-galactosamine (hereinafterabbreviated into “D-GalN”) and lipopolysaccharide (hereinafterabbreviated into “LPS”) are used in order to develop a liver diseasetherapeutic caused by virus.

Especially, since the above liver damage model induced with D-GalN/LPScauses liver damage by the immune reaction which is actually proceededin most liver diseases, it is the animal model appropriate for thetreatment and prevention of liver diseases [Ken-Ichiro Kosai, KunioMatsumoto, Hiroshi Funakoshi and Toshikazu Nakamura, Hepatocyte Growthfactor Prevents Endotoxin-induced Lethal Hepatic Failure in Mice.Hepatology, 1999, 30, 151-159]. In acute hepatitis model induced withD-GalN/LPS, D-GalN inhibits RNA synthesis and protein synthesis in cellsto maximize liver toxicity caused by LPS, and LPS promotes the secretionand synthesis of cytokine, nitrogen monoxide (NO) and active oxygen ofthe kupffer cell, which is the macrophage of liver. It has been foundout that tumor necrosis factor alpha (TNF-α) induced by excessivenitrogen monoxide is a main etiological agent of septicemia or acutehepatitis. In fact, it has been found out that TNF-α causes in vivo andin vitro hepatocyte death [Michael D Josephs, F. Rena Bahjat, KunitaroFukuzuka, Riadh Ksontini, Carmen C. Solorzano, Carl K. Edwards III,Cynthia L. Tannahill, Sally L. D. MacKAY, Edward M. Copeland III, andLyle L. Moldawer. Lipopolysaccharde and D-galactosamine-induced hepaticinjury is mediated by TNF-a and not by Fas ligand. Am J PhysiolRegulatory Integrative comp Physiol, 2000, 278, R1196-R1201]. Further,Leist considers TNF-α to be the most important factor in causing liverdamage by proving that the mortality is decreased when the acute liverdamage model induced with D-GalN/LPS is treated with anti-tumor necrosisfactor alpha (anti-TNF-α) [Leist M. Gauntner F., Bohlinger I, Germann PG, Tiegs G, Wendal A. Murine hepatocytee apoptosis induced in vitro andin vivo by TNF-α requires transcriptional arrest. J. Immunol. 1994, 153,1778-1788].

The cell death process is largely affected by Bcl-2 family (pro- andanti-apoptotic member) proteins, which can be exemplified by Bax proteinor Bid protein [Yongge Zhao, Shuchen Li, Erin E. Childs, Diane K.Kuharsky, and Xiao-Ming Yin. Activation of Pro-death Bcl-2 FamilyProteins and Mitochondria Apoptosis Pathway in Tumor NecrosisFactor-a-induced Liver Injury. J. Biol. Chem. 2001, 276, 27432-27440].

More particularly, the death process of hepatocyte activates caspase 8by interacting with FADD or TRADD protein having a death domain, whenproteins inducing apoptosis such as TNF bind to the cell receptor, TNFreceptor 1. The thus activated caspase 8 cleaves Bid protein andtransforms it into an activated form, tBid. The thus transformed tBid istranslocated to mitochondria to cause cytchrome C release. The thusreleased cytchrome C activates pro-caspase 9 into caspase 9, and thiscaspase 9 induces the cooperative effects of lower caspases byactivating caspase 3 which leads all cells to apoptosis [Xiao-Ming Yin,Bid, a critical mediator for apoptosis induced by the activation ofFas/TNF-R1 death receptors in hepatocytes. J. Mol, 2000, 78, 203-211].

Therefore, hepatocyte apoptosis generated in acute liver-injury modelinduced with D-GalN/LPS causes the activation of apoptosis pathway byTNF-α receptor. Accordingly, it can be proved that an extract of thestem or root of Acanthopanax koreanum activates liver protection by thesaid working by examining whether Acanthopanax koreanum polysaccharideinhibits TNF-α activity itself, and by proving that Acanthopanaxkoreanum polysaccharide inhibits the expression of the important proteinactivated by TNF-α.

In addition, the amount of circulating alanine aminotransferase(hereinafter abbreviated into ALT, GPT index) and aspartateaminotranasferase (hereinafter abbreviated into AST, GOT index), and theconcentration of circulating tumor necrosis factor (TNF-α) are measuredto determine liver protection activity in the acute liver damage modelinduced with D-GalN/LPS. In addition, the liver protection effect of thesample can be determined more precisely by measuring apoptosisinhibition effect of hepatocyte using the activity inhibiting hepatocyteDNA cleavage as an index, and by measuring the 24 hour survival rate ofthe mouse.

Recently a drug for the treatment or prevention of hepatitis byprotecting the liver functions by using the said animal model is beingdeveloped. Especially, it has been reported that saponin, bupleurosidecompounds (H. Maysuda et al., Bioorg. Med. Chem., 1997, 7, 2193-2198),naringin (K. Kawaguchi et al., Eur. J. Pharmacol., 1999, 368, 245-250),green tea extract(P. HE et al., J. Nutr., 2001, 131, 1560-1567),polysaccharides extracted from the seeds of Celosia argentea showactivity in protecting the liver functions in the liver damage modelinduced with D-GalN/LPS and inhibiting the experimental animal lethality(K. Hase et al., Biol, Pharm. Bull., 1996, 19, 567-572).

In addition, there is a report on the liver protection activity of anextract of Acanthopanax senticosus [Chun-Ching Lin and Pei-Chen Huang,Phytotherapy Research, 2000, 14, 489-494]. However, no specific exampleson an extract of Acanthopanax koreanum for the treatment or preventionof hepatitis or liver protection have been reported. Acanthopanaxsenticosus morphologically differs a lot from Acanthopanax koreanum.Acanthopanax senticosus is thickly wooded with thin long thorns on itsbark and branches, and the style of the fruit is divided into 5.Further, it is mainly distributed in the alpine regions of Korea;Hokkaido, Japan; the Heilung Riverside, China; and Siberia, Russia.Acanthopanax koreanum is wooded with triangle shaped grayed-brown thornswith a large base, and the style of its fruit is divided into 2.Further, it is a Korean indigenous plant distributed in the southernpart of Korea including Chejudo.

Further, Acanthopanax koreanum comprises acanthoic acid;pimara-9(11)-dien-19 oic acid as its main component, whereas,Acanthopanax senticosus does not comprise such component [Young H. Kimand Bo S. Chung J. Nat. Prod. 1988, 51 1080-1083].

Therefore, in connection with liver diseases caused by hepatitis virusand toxic substances, inventors have devoted themselves in developing aliver disease therapeutic with less side effects, and based on theworking model of the acute liver-injury induced with D-GalN/LPS thepresent invention has been completed by proving an accurate experimentalmethod and results that an extract from the root or stem of Acanthopanaxkoreanum is effective on the treatment and prevention of liver damagewhile maintaining the structure and function of the liver tissues.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an extract ofAcanthopanax koreanum for the treatment or prevention of hepatitis orthe liver protective drug.

It is another object of the present invention to provide a use whereinan extract of Acanthopanax koreanum can be used for the treatment orprevention of hepatitis or as a liver protective drug on the basis ofthe working model of the acute liver-injury mice induced withD-GalN/LPS.

BEST MODE FOR CARRYING OUT THE INVENTION

FIG. 1 shows HPLC analysis of the fraction containing polysaccharidewith a molecular weight larger than range of 12,000˜14,000, among theethanol insoluble part, obtained from the root of Acanthopanax koreanum

FIG. 2 shows HPLC analysis of the fraction containing polysaccharidewith a molecular weight larger than range of 12,000˜14,000, among theethanol insoluble part, obtained from the stem of Acanthopanax koreanum.

FIG. 3 shows effect of the extract obtained from the root ofAcanthopanax koreanum, on the survival of mice in liver-injury modelinduced by D-GalN/LPS.

a group treated with the water extract of Acanthopanax koreanum root(hereinafter abbreviated into “SRW”, 300 mg/kg);

a group treated with the 80%-ethanol insoluble part of the water extractof Acanthopanax koreanum root (hereinafter abbreviated into “SRWB”, 300mg/kg); and

● a group treated with physiological saline solution

FIG. 4 shows effect of the extract obtained from the stem ofAcanthopanax koreanum, on the survival of mice in liver-injury modelinduced by D-GalN/LPS

a group treated with the water extract of Acanthopanax koreanum stem(hereinafter abbreviated into “SSW”, 300 mg/kg);

a group treated with the 80%-ethanol insoluble part of the water extractof Acanthopanax koreanum stem (hereinafter abbreviated into “SSWB”, 300mg/kg); and

● a group treated with physiological saline solution

FIG. 5 shows effect of the fraction containing polysaccharide with amolecular weight larger than 100,000, among the said 80%-ethanolinsoluble part obtained from the stem of Acanthopanax koreanum, on thesurvival of mice in liver-injury model induced by D-GalN/LPS.

▪ a group treated with the fraction containing polysaccharide with amolecular weight larger than 100,000, obtained from the stem ofAcanthopanax koreanum (30 mg/kg);

▴ a group treated with the fraction containing polysaccharide with amolecular weight larger than 100,000, obtained from the stem ofAcanthopanax koreanum group (100 mg/kg); and

♦ a group treated with physiological saline solution

FIG. 6 shows effect of the fraction containing polysaccharide with amolecular weight larger than 100,000, among the said 80%-ethanolinsoluble part obtained from the root of Acanthopanax koreanum on thesurvival of mice in liver-injury model induced by D-GalN/LPS.

□ a group treated with the fraction containing polysaccharide with amolecular weight larger than 100,000, obtained from the root ofAcanthopanax koreanum (30 mg/kg)

▴ a group treated with the fraction containing polysaccharide with amolecular weight larger than 100,000, obtained from the root ofAcanthopanax koreanum (100 mg/kg);and

♦ a group treated with physiological saline solution

FIG. 7 shows effect of the extract obtained from the root or stem ofAcanthopanax koreanum, on the survival of mice in liver-injury modelinduced by D-GalN/TND-α.

□ a group treated with the fraction containing polysaccharide with amolecular weight larger than 100,000, obtained from the stem ofAcanthopanax koreanum (100 mg/kg)

▴ a group treated with the fraction containing polysaccharide with amolecular weight larger than 100,000, obtained from the root ofAcanthopanax koreanum (100 mg/kg);and

♦ a group treated with physiological saline solution

FIG. 8 shows effect of the extract on the DNA fragmentation of livercell in liver-injury model induced by D-GalN/LPS.

Marker: comparative marker

1: DNA isolated from the liver of the mice to which physiological salinesolution was administered.

2: DNA isolated from the liver of the mice to which physiological salinesolution was administered after administration of D-GalN/LPS.

3: DNA isolated from the liver of the mice to which D-GalN/LPS and 300mg/kg of the water extract obtained from the root of Acanthopanaxkoreanum were administered.

4: DNA isolated from the liver of the mice to which D-GalN/LPS and 300mg/kg of the water extract obtained from the stem of Acanthopanaxkoreanum were administered.

5: DNA isolated from the liver of the mice to which D-GalN/LPS and 300mg/kg of the 80%-ethanol insoluble part of the water extract obtainedfrom the stem of Acanthopanax koreanum were adminstered.

6: DNA isolated from the liver of the mice to which D-GalN/LPS and thefraction containing polysaccharide with a molecular weight larger thanrange of 12,000˜14,000, obtained by dialyzing the said 80%-ethanolinsoluble part were administered.

FIG. 9 shows effect of the fraction containing polysaccharide with amolecular weight larger than 100,000, among the ethanol insoluble parton the expression of pro-apoptotic protein;

A: the extract obtained from the stem of Acanthopanax koreanum

B: the extract obtained from the root of Acanthopanax koreanum

FIG. 10 shows effect of the oral administration of the 80%-ethanolinsoluble part of the water extract obtained from the stem ofAcanthopanax koreanum on the liver protection.

FIG. 11 shows effect of the oral administration of the ethanol insolublepart of the water extract obtained from the root of Acanthopanaxkoreanum on the liver protection.

▴: a group treated with the 80%-ethanol insoluble part of the waterextract obtained from the stem or root of Acanthopanax koreanum (30mg/kg)

: a group treated with the 80%-ethanol insoluble part of the waterextract obtained from the stem or root of Acanthopanax koreanum (100mg/kg);and

♦ a group treated with physiological saline solution

The present invention provides an extract of Acanthopanax koreanum forthe treatment or prevention of hepatitis, or as a liver protective drug.

The extract of the present invention includeds 1) the water extract ofthe root or stem of Acanthopanax koreanum 2) the ethanol insoluble partamong the said water extract, obtained by treating the said waterextract with ethanol, 3) the fraction containing polysaccharide with amolecular weight larger than range of 12,000˜14,000, among the saidethanol insoluble part, or 4) the fraction containing polysaccharidewith a molecular weight larger than 100,000, among the said ethanolinsoluble part.

Furthermore, the present invention provides a use of the extractobtained from the root or stem of Acanthopanax koreanum, as atherapeutic agent for treatment or preventer of hepatitis, or a liverprotective drug.

The extract of the present invention inhibits activity of TNF-α andexpression of important protein activated by TNF-α in acute liver-injurymice model induced by D-GalN/LPS. Also, the extract of the presentinvention made high survival rate maintained in the experiment formeasurement of lethality rate progressed for 24 hours. Therefore, basedon the working model of the acute liver-injury mice induced byD-GalN/LPS, the extract of the present invention can be used as atherapeutic agent or preventer of the hepatitis, or a liver protectivedrug.

The present invention uses Acanthopanax koreanum of which the place oforigin is KOREA. The known extract of Acanthopanax sp. relates to theextract of the stem or leaves of Acanthopanax senticosus. Furthermore,to apply Acanthopanax senticosus to experimental or drug material, itmust be withered up. However, in the present invention, the extract ofAcanthopanax koreanum obtained from the root of the Acanthopanaxkoreanum has the same efficiency as one obtained from the stem thereof.Therefore, Acanthopanax koreanum does not have to be withered up.

The said water extract of Acanthopanax koreanum is prepared by treatingthe root or stem of Acanthopanax koreanum with water by the method suchas dipping, maceration or heating. Preferably, the said water extract isprepared by heating the root or stem of Acanthopanax koreanum at thetemperature higher than 90° C.

The ethanol insoluble part of the said water extract is prepared bytreating the said water extract of Acanthopanax koreanum with ethanol,of which the final concentration of ethanol is 50˜90%. Preferably, theconcentration is 75˜85%, more preferably 80%.

The fraction containing polysaccharide with a molecular weight largerthan range of 12,000˜14,000, or larger than 100,000, among the saidethanol insoluble part is prepared by dialysis of the said extractthrough dialyzing diaphagram or filter membrane corresponding to themolecular weight, respectively.

As shown in the experiment using liver injury mice model induced byD-GalN/LPS, the said water extract of Acanthopanax koreanum of thepresent invention, preferably the said ethanol insoluble part, morepreferably the fraction containing polysaccharide with a molecularweight larger than range of 12,000˜14,000, or larger than 100,000, amongthe said ethanol insoluble part has an excellent efficiency for thetreatment or prevention of hepatitis, or a liver protective drug.

FIG. 3 or 4 shows the lethality rate of the mice induced by D-GalN/LPS,measured for 24 hours when the extract of Acanthopanax koreanum isadministered to the said mice. More particularly, the said water extractand the said 80%-ethanol insoluble part shows excellent survival rate bymore than 70˜80% in 24 hours without liver toxicity. Therefore, theextract has a high efficacy such as liver protection in the mice modelof the acute hepatitis. Also, FIG. 5 and FIG. 6 show that the fractioncontaining polysaccharide with a molecular weight larger than 100,000,among the ethanol insoluble part obtained from the root or stem ofAcanthopanax koreanum maintains the survival rate higher than 90%,respectively. Especially, the fraction obtained from the root ofAcanthopanax koreanum has an efficiency that when 30 mg/kg or 100 mg/kgof the said fraction was administered to the mice, the survival rate ismaintained to the extent of 100%. Therefore, the fraction has anexcellent efficacy for the liver protection.

FIG. 8 shows that the said ethanol insoluble part prepared by treatingthe said water extract with ethanol, of which the final concentration ofethanol is 80 and the fraction containing polysaccharide with amolecular weight larger than range of 12,000˜14,000, among the ethanolinsoluble part inhibit DNA fragmentation in liver cell.

FIG. 9 shows that the fraction containing polysaccharide with amolecular weight larger than 100,000, among the said ethanol insolublepart influences the expression of pro-apoptotic protein. Particularly,the result shows that the said fraction used in the experiment inhibitsthe expression of Bax protein concentration dependently. Also, in caseof Bid protein which is one of pro-apoptotic proteins, experimentalgroup to which the fraction containing polysaccharide with a molecularweight larger than 100,000 is administered, has a similar inhibitionrelative to the control group concentration dependently. Therefore, thesaid fraction containing polysaccharide with a molecular weight largerthan 100,000 inhibits the expression of pro-apoptotic protein activatedby TNF-α in liver cell.

Also, FIG. 10 and FIG. 11 show that when the 80%-ethanol insoluble partprepared by treating the water extract with ethanol is orallyadministered to the mice, the survival rate of the mice is maintained tothe extent of 50% for 24 hours. However, the control group to which thesaid part is not administered is deceased within 12 hours.

Therefore, based on the working model of the acute liver-injury miceinduced by D-GalN/LPS, the extract of the present invention maintainsthe serum level of TNF-α, the cause of acute hepatitis, to be similar tolevel of the normal group. Also, the said extract inhibits theexpression of pro-apoptotic protein activated by TNF-α. Also, the saidextract reduces the serum level of AST or ALT to the similar to that ofnormal group. Therefore, the said extract has an excellent efficacy fortreatment or prevention of hepatitis, or a liver protection.

The extract of Acanthopanax koreanum of the present invention can beused for health supplementary food.

The extract of the present invention can be administered in variousmethods in an amount of dosage. The said composition containspharmaceutically acceptable carrier. More particularly, any ofpharmaceutically acceptable carrier selected from sterilized solution,or the standard carrier used in the known formulation, such as tablet,coating tablet and capsule can be used. Conservatively, carrier isselected from diluting agent containing starch, milk, glucose, clay,gelatin, stearic acid, talc, vegetable oil, gum and glycol, or the knowndiluting agents. Also, the said carrier can be selected from flavoringagent, pigment or another components.

To administer pharmaceutical composition containing the extract of thepresent invention as an effective ingredient within the said dosage,administrations can be accomplished by the conservative methods such asoral administration, intravenous injection, intramuscular injection ortransdermal administration, but is not limited to them. In clinicalapplication, the formulation can be administered through the oral orparenteral administration. The said formulation is prepared by usingcommon diluent containing packing agent, bulking agent, bindng agent,moistening agent, disintegrating agent, surfactant or diluting agent.

Composition for oral administration can be formulated into the solidformulation such as tablet, pill, powder, granule or capsule. The saidformulation is prepared by mixing one or more selected from the extractof the present invention with diluting agent for example, starch,calcium carbonate, sucrose, lactose or gelatin. Also, in addition tosimple diluting agent, lubricant such as magnesium stearate is used.

Composition for oral administration can be formulated into the liquidformulation such as suspension, liquid, emulsion, syrup. The saidformulation contains various diluting agent for example, moisteningagent, flavoring agent, aromatic agent or preservative, in addition tosimple diluent, for example water, liquid paraffin. Preferably, thecomposition can be formulated into the tablet, capsule, or drink, andused as medicine or health supplementary food.

Also, the pharmaceutical composition of the present invention can beparenterally administered. The formulation for parenteral administrationis prepared by mixing one or more selected from the extract of thepresent invention with stabilizer or buffer in water, preparing intosolution or suspension, and formulating into an unit dosage form such asample or vial.

The extract used for therapeutic agent or preventor of hepatitis, or aliver protective drug is preferably the ethanol insoluble part, morepreferably the fraction containing polysaccharide with a molecularweight larger than range of 12,000˜14,000 among the said ethanolinsoluble part. However, in the process of purification using dialyzingdiaphagram, yield is 30˜40%. Therefore, preferably, the ethanolinsoluble part is used. An effective ingredient of the present inventioncan be administered through one or many times per 1 day. The dosage ofthe effective ingredient is preferable 1˜1000 mg/kg/day, more preferable10˜1000 mg/kg/day, depending upon absorptivity of the active componentin vivo, activity, excretion rate, age, sex and state of the patients,seriousness of disease under treatment. More particularly, thepreferable dosage of the water extract, the 70%-ethanol insoluble part,and the fraction containing polysaccharide with a molecular weightlarger than range of 12000˜14000, among the said ethanol insoluble partis 300˜1000 mg/kg/day, 100˜500 mg/kg/day and 10˜300 mg/kg/day,respectively. Accurate dosage, method and frequency for administrationwere selected according to property of the formulation, weight and stateof administrative group, or characteristic of derivatives used.

Toxic test for the mice shows that the extract of the present invention,obtained from the root or stem of Acanthopanax koreanum is nontoxic.Also, the test shows that LD50 is higher than 2000 mg/kg, the extract ofthe present invention has high safety and stability. Therefore, theextract of the present invention, as a liver protective drug can besafely administered to the body.

The present invention will be explained in more detail with reference tothe following examples. However the following examples are provided onlyto illustrate the present invention, and the present invention is notlimited to them.

EXAMPLE 1

Preparation of the Water Extract of Acanthopanax koreanum

The root or stem of Acanthopanax koreanum is dried and sliced to smallpieces, respectively. In a 10 L flask, 1 kg of slices was mixed withwater and extracted at the temperature more than 90° C. for 3 hours. Theextraction is repeated two times. The extract is filtered through thefiltrate memberane, concentrated under reduced pressure and lyophilizedto prepare the water extract. Quantity of the water extract obtainedfrom the root or stem of Acanthopanax koreanum is 142 g or 80 g,respectively.

EXAMPLE 2

Preparation of the Ethanol Insoluble Part of Acanthopanax koreanum

The ethanol insoluble part containing more polysaccharide relative tothe said water extract was prepared by treating the said water extractwith ethanol. 20 g of the said water extract obtained from the root orstem of Acanthopanax koreanum was dissolved in 50 ml of water. Ethanolwas added to the solution, of which final concentration of ethanol was60˜80%. The solution was centrifuged to acquire the precipitatedinsoluble part. The insoluble part was dried.

Also, the ethanol filtrate after centrifugal separation was concentratedto acquire the ethanol extract from the said water extract obtained fromthe root or stem of Acanthopanax koreanum, respectively. Quantity of theethanol insoluble part and ethanol extract obtained by treating thewater said extract of the root or stem of Acanthopanax koreanum withethanol was shown in the table 1.

TABLE 1 Quantity of the ethanol insoluble part and the ethanol extractFinal Quantity of Conc. of the water Root Stem ethanol extract(g)SRWB^(a)(g) SRWS^(b)(g) SSWB^(c)(g) SSWS^(d)(g) 60% 20 4.5 15.3 3.7 16.570& 20 6.9 13.5 5.1 15.2 80% 20 8.6 11.4 5.6 14.9 ^(a)the ethanolinsoluble part of the water extract obtained from the root ofAcanthopanax koreanum(hereinafter, abbreviated into SRWB) ^(b)theethanol soluble part of the water extract obtained from the root ofAcanthopanax koreanum(hereinafter, abbreviated into SRWS) ^(c)theethanol insoluble part of the water extract obtained from the stem ofAcanthopanax koreanum(hereinafter, abbreviated into SSWB) ^(d)theethanol soluble part of the water extract obtained from the stem ofAcanthopanax koreanum(hereinafter, abbreviated into SSWS)

As shown in the table 1, in case of treating the water extract withethanol, the yield of ethanol insoluble part was higher when finalconcentration of ethanol is 80% than when final concentration of ethanolis 60% or 70%.

EXAMPLE 3

Preparation of the Fraction Containing Polysaccharide with a MolecularWeight Larger than Range of 12,000˜14,000, Among the said EthanolInsoluble Part

The ethanol insoluble part obtained by treating the water extract withethanol has polysaccharide as the major component. However, to purifythe said polysaccharide, the fraction containing polysaccharide with amolecular weight larger than range of 12000˜14000 is prepared bydialysis of the said extract through dialyzing diaphagram (Spectra PorSpectrum Medical Industries Inc. Houston Tex.) corresponding to themolecular weight, respectively.

The water extract obtained from the root or stem of Acanthopanaxkoreanum was treated with 80% ethanol to prepare the ethanol insolublepart, respectively. The 500 mg of the said ethanol insoluble part wasdissolved in 7 ml of distilled water, thereafter the solution wascentrifuged to acquire the supernatant. The said supernatant wasdialysed through the dialyzing diaphagram passing the compound with amolecular weight in the range of 12000˜14000, thereafter filter cake waslyophilized. Quantity of the fraction containing polysaccharide with amolecular weight larger than range of 12000˜14000, obtained from the 300mg of the ethanol insoluble part prepared from the root or stem ofAcanthopanax koreanum is 130 mg or 120 mg, respectively.

EXAMPLE 4

Preparation of the Fraction Containing Polysaccharide with a MolecularWeight Larger than 100,000, Among the said Ethanol Insoluble Part

To purify the polysaccharide of Acanthopanax koreanum additionally, thefraction containing polysaccharide with a molecular weight larger than100,000 is prepared by filtering the said extract through dialyzingdiaphagram (Spectra Por Spectrum Medical Industries Inc. Houston Tex.)passing the compound with a molecular weight smaller than 100,000.

The water insoluble part obtained from the root or stem of Acanthopanaxkoreanum was treated with ethanol to prepare the ethanol extract. 15.5 gor 20.7 g of the ethanol insoluble part was dissolved in the water,respectively. The solution was dialysed through the dialyzing diaphagrampassing the compound with a molecular weight smaller than 100,000,thereafter filter cake was lyophilized. Yield of the fraction containingpolysaccharide with a molecular weight larger than 100,000, obtainedfrom the ethanol insoluble part prepared from the root or stem ofAcanthopanax koreanum is 12% or 10.2%, respectively.

The experiment confirming that the extract of the present invention hasefficiency for treatment of hepatitis or liver protection inliver-injury mice model induced by D-GalN/LPS was accomplished as shownin the below.

EXPERIMENTAL EXAMPLE 1

HPLC Analysis of the Fraction Containing Polysaccharide

As for the fraction containing polysaccharide with a molecular weightlarger than range of 12,000˜14,000, obtained from the said ethanolinsoluble part, molecular weight or number of the polysaccharide wasmeasured.

The fraction containing polysaccharide with a molecular weight largerthan range of 12,000˜14,000, obtained from the root or stem ofAcanthopanax koreanum was dissolved in the distilled water, of which thefinal concentration of polysaccharide was 10 mg/ml. 20 μl of thesolution was injected to YMC-pack Diol-300 column (YMC Co. Kyoto,Japan). The elution rate of eluent is 1 ml/min. Molecular weight andnumber of the polysaccharide in the sample were analyzed by evaporationlight scattering detector (Alltech 500 ELSD) (shown in FIG. 1 or FIG.2).

The extract containing polysaccharide, obtained from the stem ofAcanthopanax koreanum is mainly comprised of polysaccharide with amolecular weight 900,000. Also, polysaccharide with a various molecularweight, for example, 450,000 or 250,000 was contained in the extract.Also, polysaccharide with a molecular weight in the range of14,000˜200,000 was contained in the extract. The extract containingpolysaccharide, obtained from the root of Acanthopanax koreanum ismainly comprised of polysaccharide with a molecular weight more than1,000,000. Also, polysaccharide with a various molecular weight, forexample, 2,000,000, 450,000 or 300,000 was contained in the extract.More polysaccharide with a molecular weight in the range of14,000˜200,000 was contained in the extract of the root than that of thestem.

EXPERIMENTAL EXAMPLE 2

The Serum Level of AST or ALT in Liver-Injury Mice Model Induced byD-GalN/LPS

The experiment confirming that the extract of the present invention hasa efficacy for prevention of hepatitis in liver-injury mice modelinduced by D-GalN/LPS by measuring the serum level of AST or ALT wasaccomplished as shown in the below.

After mice (B57BL/6) having body weights of 20 g were adjusted to a newenvironment for 1 week, they were used in the experiment. Mice werefully fed before the experiment. The experiment was accomplished withmice divided to three groups such as normal group, group to whichphysiological saline solution was administered (physiological salinesolution-treated group), group to which the extract was administered(theextract-treated group). The said extract was selected from the groupconsisting of 1) the methanol extract obtained by treating the root orstem of Acanthopanax koreanum with 100% methanol, 2) the water extractof Acanthopanax koreanum obtained by treating the root or stem ofAcanthopanax koreanum with water, 3) the ethanol extract obtained bytreating the root or stem of Acanthopanax koreanum with 70% ethanol 4)the ethanol insoluble part among the said water extract, obtained bytreating the said water extract with ethanol, of which finalconcentration of ethanol is 80%, or 5) the fraction containingpolysaccharide with a molecular weight larger than range of12,000˜14,000 among the ethanol insoluble part.

The said extract dissolved in physiological saline solution wasintraperitoneally administered to the said mice model twice in an amountof 50 mg/kg or 300 mg/kg, respectively, at 12 and 1 hour earlier thanD-Gal and LPS treatment. Thereafter, D-Gal and LPS were subsequentlyadministered to the said mice model in an amount of 700 mg/kg and 10mg/kg, respectively.

Physiological saline solution was administered to the control group ofthe said mice model in the same amount. Blood was collected from themice at 8 hours after administration. DNA was isolated from the part ofliver tissue. The part of liver was maintained in 10% formalin forstaining of tissue. The collected blood was centrifuged at 3000 rpm, theserum was isolated and stored at −20° C. The serum level of AST and ALTwas measured by using kit purchased from the ARKARY FACTORY (JAPAN) andauto-dry chemistry analyzer (SPOTCHEM, ARKARY, JAPAN) to measure theserum level of GOT and GPT in blood. The result was shown in table 2 and3.

TABLE 2 The effect of the various root extract of Acanthopanax koreanumon the serum level of AST and ALT in liver-injury mice model induced byD-GalN/LPS Amount Group (mg/kg) AST ALT Normal group — 67 ± 11 23 ± 11Physiological saline — 3456 ± 1064 3678 ± 1291 solution-treated group(control group) The water extract-treated 50 567 ± 181 548 ± 139 group300 190 ± 40  117 ± 47  The 70% ethanol extract- 50 678 ± 125 598 ± 217treated group 300 228 ± 45  96 ± 33 Treatment The ethanol 50 378 ± 113364 ± 128 of the insoluble part- 300 189 ± 45  143 ± 53  water treatedgroup extract The ethanol 50 589 ± 139 543 ± 145 with soluble part- 300450 ± 120 425 ± 107 ethanol treated group The fraction containing 50 127± 35  118 ± 31  polysaccharide with a molecular weight larger than rangeof 12,000~14,000 The fraction containing 30 125 ± 32  97 ± 35polysaccharide with a 100 92 ± 25 76 ± 30 molecular weight larger than100,000 The said result derives from mean ± SEM of measurements obtainedfrom the experiments of five times, five times and six times as fornormal group, saline solution-treated group and the extract-treatedgroup, respectively.

TABLE 3 The effect of the various stem extract of Acanthopanax koreanumon the serum level of AST and ALT in liver-injury mice model induced byD-GalN/LPS. Amount Group (mg/kg) AST ALT Normal group — 67 ± 11 23 ± 11Physiological saline — 3456 ± 1064  3678 ± 1291* solution-treatedgroup(control group) The water extract-treated 50 598 ± 194 634 ± 157group 300 165 ± 13  26 ± 13 The 70% ethanol extract- 50 2987 ± 1859 3125± 1032 treated group 300 3283 ± 1959 3457 ± 2373 Treatment The ethanol50 446 ± 115 464 ± 167 of the insoluble part- 300 131 ± 11  19 ± 11water treated group extract The ethanol 50 550 ± 104 567 ± 134 withsoluble part- 300 523 ± 130 543 ± 145 ethanol treated group The fractioncontaining 50 121 ± 14  26 ± 11 polysaccharide with a molecular weightlarger than range of 12,000~14,000 The fraction containing 30 112 ± 15 32 ± 12 polysaccharide with a 100 91 ± 12 21 ± 15 molecular weightlarger than 100,000 The said result derives from mean ± SEM ofmeasurements obtained from the experiments of five times, five times andsix times as for normal group, saline solution-treated group and theextract-treated group, respectively.

As shown in the table 2 and 3, the serum level of AST and ALT in micemodel, which administered only saline with D-GalN/LPS was markedlyincreased relative to that of the normal group.

Also, the serum level of AST and ALT in mice model, which administeredthe 70% ethanol extract or the supernatant prepared by treating thewater extract with 80% ethanol was similar to that of the physiologicalsaline solution-treated group. Therefore, it was shown that the 70%ethanol extract and the supernatant have no efficiency for the treatmentof hepatitis.

However, the serum level of AST and ALT in mice model, whichadministered 50 mg/kg of the water extract was decreased greatlyrelative to that of the physiological saline solution-treated group. Theserum level of AST and ALT in mice model, which administered the80%-ethanol insoluble part was more decreased. More preferably, theserum level of AST and ALT in mice model, which administered 50 mg/kg ofthe fraction containing polysaccharide with a molecular weight largerthan range of 12,000˜14,000, among the ethanol insoluble part was thelowest one of all, and was similar to the that of normal group. The saidfraction has excellent efficacy for treatment of hepatitis.

EXPERIMENTAL EXAMPLE 3

The Serum Level of TNF-α of the Extract on the Liver-Injury Mice ModelInduced by D-GalN/LPS

The experiment measuring the serum level of TNF-α, direct mediator ofacute hepatitis in liver-injury mice model induced by D-GalN/LPS wasaccomplished as shown in the below.

After mice (B57BL/6) having body weights of 20 g were adjusted to a newenvironment for 1 week, they were used in experiment. Mice were fullyfed before the experiment. The experiment was accomplished with micedivided to three groups such as normal group, group to whichphysiological saline solution was administered (physiological salinesolution-treated group), group to which the extract was administered(the extract-treated group). The extract was administered to the mice inthe same method as the experimental example 2.

The said extract dissolved in physiological saline solution wasintraperitoneally administered to the mice in an amount of 50 mg/kg or300 mg/kg for two times, respectively. Thereafter D-Gal and LPS weresubsequently administered to the mice in an amount of 700 mg/kg and 10mg/kg.

Physiological saline solution was administered to the mice in the sameamount. Blood was collected from mice at 1 hour after administration.The collected blood was kept at the room temperature for 1 hour andcentrifuged, the serum was isolated. The prepared serum and liver werestored at −20° C.

The serum level of TNF-α was measured by using enzyme-linkedimmunosorbent assay (ELISA) kit. The result was shown in table 4 and 5.

TABLE 4 The effect of various extracts of Acanthopanax koreanum root onthe serum level of TNF-α in liver-injury mice model induced byD-GalN/LPS. Group Amount(mg/kg) TNF-α (pg/ml) Normal group — 26 ± 13Physiological saline solution- — 678 ± 29  treated group(control group)The water extract-treated group 50 124 ± 36  300 74 ± 26 The 70% ethanolextract-treated 50 648 ± 104 group 300 587 ± 87  Treating the Theethanol 50 102 ± 26  water extract insoluble part- 300 59 ± 15 withethanol treated group The ethanol 50 605 ± 92  soluble part- 300 260 ±45  treated group The fraction containing 50 32 ± 12 polysaccharide witha molecular weight larger than range of 12,000~14,000 The fractioncontaining 30 45 ± 21 polysaccharide with a molecular 100 29 ± 12 weightlarger than 100,000 The said result derives from mean ± SEM ofmeasurements obtained from the experiments of five times, five times andsix times as for normal group, physiological saline solution-treatedgroup and the extract-treated group, respectively.

TABLE 5 The effect of various extracts of Acanthopanax koreanum stem onthe serum level of TNF-α in liver-injury mice model induced byD-GalN/LPS. Group Amount(mg/kg) TNF-α (pg/ml) Normal group — 26 ± 11Physiological saline solution- — 785 ± 17  treated group(control group)The water extract-treated group 50 132 ± 28  300 67 ± 16 The 70% ethanolextract-treated 50 690 ± 110 group 300 678 ± 54  Treating the Theethanol 50 62 ± 16 water extract insoluble part- 300 566 ± 55  withethanol treated group The ethanol 50 233 ± 43  soluble part- 300 28 ± 11treated group The fraction containing 50 32 ± 12 polysaccharide with amolecular weight larger than range of 12,000~14,000 The fractioncontaining 30 32 ± 12 polysaccharide with a molecular 100 25 ± 11 weightlarger than 100,000 The said result derives from mean ± SEM ofmeasurements obtained from the experiments of five times, five times andsix times as for normal group, saline solution-treated group and theextract-treated group, respectively.

As shown in the table 4 and 5, the serum level of TNF-α in mice to whichD-GalN/LPS was only administered except the extract was increased thirtytimes as much as that of the normal group.

The serum level of TNF-α in mice to which 300 mg/kg of the water extractwas administered was decreased.

More preferably, the serum level of TNF-α in mice to which the80%-ethanol insoluble part of the water extract was administered was themost similar to that of normal group. The said 80%-ethanol insolublepart has an excellent efficacy for treatment of hepatitis.

EXPERIMENTAL EXAMPLE 4

Effect of the Extract on the Survival of the Liver-Injury Mice ModelInduced by D-GalN/LPS

The experiment measuring survival of liver-injury mice model induced byD-GalN/LPS was accomplished for 24 hours as shown in the below.

After mice (B57BL/6) having body weights of 20 g were adjusted to a newenvironment for 1 week with normal diet, they were used in experiment.Mice were starved for twenty hours before the experiment. Thereafter,700 mg/kg of D-Gal and 10 mg/kg of LPS were administered to the mice.Liver-injury mice model induced by D-GalN/LPS was prepared for theexperiment. The experiment was accomplished with mice divided to threegroups such as normal group, group to which saline solution wasadministered, group to which the extract was administered. The saidextract is the water extract obtained by treating the root or stem ofAcanthopanax Koreanum with water, the 80%-ethanol insoluble part amongthe said water extract, or the fraction containing polysaccharide with amolecular weight larger than 100,000 among the said 80%-ethanolinsoluble part.

Physiological saline solution was administered to the mice in the sameamount.

1. The Effect of the said Water Extract or the said 80%-EthanolInsoluble Part on the Lethality Rate of the Mice.

The said water extract or the said 80%-ethanol insoluble part dissolvedin physiological saline solution was administered to the mice in anamount of 50 mg/kg or 300 mg/kg, respectively. Thereafter, lethalityrate was measured for 24 hours after administration.

FIG. 3 shows that the water extract obtained from the root ofAcanthopanax koreanum decreased the lethality of the mice induced byD-GalN/LPS. Group to which physiological saline solution wasadministered was diseased at 6 hours after D-GalN/LPS. All of the group(eight mice) was diseased within 24 hours.

However, seven of ten mice to which 300 mg/kg of the water extract wasadministered survived within 24 hours, then the survival rate resultedin 80%. Nine of ten mice to which 300 mg/kg of the said 80%-ethanolinsoluble part was administered survived within 24 hours, then thesurvival rate resulted in 90%.

FIG. 4 shows that the water extract obtained from the stem ofAcanthopanax koreanum decreased the lethality of the mice induced byD-GalN/LPS. Group to which physiological saline solution wasadministered was diseased at 6 hours after D-GalN/LPS. Nine mice of thegroup (ten mice) were diseased within 24 hours.

However, seven of ten mice to which 300 mg/kg of the water extract wasadministered survived within 24 hours, then the survival rate resultedin 70%. Nine of ten mice to which 300 mg/kg of the said 80%-ethanolinsoluble part was administered survived within 24 hours, then thesurvival rate resulted in 90%.

The said result shows that the said water extract or the said ethanolinsoluble part obtained from the root or stem of Acanthopanax koreanummade the survival rate maintained in the range of 70%˜90%. Therefore,the water extract or the 80%-ethanol insoluble part has an efficacy fora liver protection.

2. The Effect of the Polysacccharide Fraction with Molecular WeightLarger than 100,000 on the Lethality Rate of the Mice.

The water extract dissolved in physiological saline solution wasintraperitoneally administered to the mice in an amount of 30 mg/kg or100 mg/kg for two times, respectively. Thereafter, lethality rate wasmeasured after administration.

700 mg/kg of D-GalN and 10 mg/kg of LPS was administered to the mice,thereafter the fraction containing polysaccharide with a molecularweight larger than 100,000, among the ethanol insoluble part wasadministered to the mice in an amount of 30 mg/kg or 100 mg/kg,respectively. Thereafter, lethality rate was measured for 24 hours afteradministration.

FIG. 5 shows that the water extract obtained from the stem ofAcanthopanax koreanum decreased the lethality rate of the mice inducedby D-GalN/LPS. Group to which physiological saline solution wasadministered was diseased at 5 hours after administration of D-GalN/LPS.Also, lethality rate reached 84% (ten of twelve mice were diseased.) at6 hours after administration. Lethality rate reached 92% (eleven oftwelve mice were diseased.) at 15 hours after administration.

However, lethality rate of mice to which 30 mg/kg or 100 mg/kg of thefraction containing polysaccharide with a molecular weight larger than100,000, obtained from the stem of Acanthopanax koreanum wasadministered reached 16% (two of twelve mice were diseased.) or 8% (oneof twelve mice were diseased.) at 24 hours after administration,respectively. Therefore, the fraction has an efficacy for a liverprotection.

As shown in FIG. 6, mice to which the said fraction was not administeredwas diseased at 5 hours after administration of D-GalN/LPS. Also,lethality rate reached 86% (six of seven mice were diseased.) at 6 hoursafter administration. Lethality rate reached 100% (all of twelve micewere diseased.) at 7 hours after administration.

However, lethality rate of mice to which 30 mg/kg or 100 mg/kg of thefraction containing polysaccharide with a molecular weight larger than100,000, obtained from the stem of Acanthopanax koreanum wasadministered reached 0% (all of twelve mice were diseased.).

EXPERIMENTAL EXAMPLE 5

Effect of the Extract on the Survival of the Liver-Injury Mice ModelInduced by D-GalN/TNF-α

To confirm whether liver protection of the Acanthopanax koreanum isdirectly connected to the inhibition of LPS-induced production of TNF-αor inhibition of signaling pathway through TNF-α receptor or not, theexperiment was accomplished at the same procedure of the said experimentwith intravenous administration of TNF-α in an amount of 15 μg/kginstead of LPS. Therefore, acute liver-injury mice model induced byD-GalN/TNF-α were prepared for the experiment.

For the experiment, the fraction containing polysaccharide with amolecular weight larger than 100,000, displaying the most excellentsurvival in the said experiment 5, was administered to the said mice. Incase of control group, physiological saline solution was onlyadministered to the mice. Six mice per group were used for theexperiment. TNF-α was intravenously administered to the mice and thenimmediately D-GalN was intraperitoneally administered.

The fraction was administered to the mice at 12 hours and 1 hour beforeadministration of D-GalN/TNF-α. The result was shown in FIG. 7. Five ofsix mice of the control group were diseased at 6 hours afteradministration of D-GalN/TNF-α, then lethality rate reached 83%.However, one of six mice of the administrative group were diseased to 8hours after administration of D-GalN/TNF-α, then lethality rate reach17%. Therefore, the fraction containing polysaccharide show the highsurvival in mice to which TNF-α was directly administered.

EXPERIMENTAL EXAMPLE 6

Measurement of DNA Fragmentation in Liver Cell

The experiment confirming whether the extract inhibits apoptosis ofliver cell in the liver-injury mice model induced by D-GalN/LPS wasaccomplished as shown in the below.

DNA was isolated from the liver tissue of the mice. Electrophoresis ofthe isolated DNA was accomplished, thereafter electro gel was dyed withethidium bromide to confirm whether DNA was fragmented or not.

FIG. 8 show that electrophoresis of DNA (3) and (4) isolated from themice to which 300 mg/kg of the water extract obtained from the root orstem of Acanthopanax koreanum was administered was similar to one of DNA(1) isolated from the mice to which physiological saline solution wasadministered. Preferably, electrophoresis of DNA (5) isolated from themice to which 300 mg/kg of the ethanol insoluble part obtained from theroot or stem of Acanthopanax koreanum was administered shows that thesaid part has an efficacy to inhibit DNA fragmentation of liver cell inthe liver-injury mice model induced by D-GalN/LPS. Furthermore, thefraction containing polysaccharide with a molecular weight larger thanrange of 12,000˜14,000 inhibits the fragmentation of DNA induced byD-GalN/LPS. Therefore, the extract of Acanthopanax koreanum of thepresent invention inhibits the fragmentation of DNA induced byD-GalN/LPS, thus has an efficacy to inhibit apoptosis of liver cell.

EXPERIMENTAL EXAMPLE 7

The Effect of the Fraction Containing Polysaccharide with a MolecularWeight Larger than 100,000 on the Expression of Pro-Apoptotic Protein.

The experiment confirming whether the fraction containing polysaccharidewith a molecular weight larger than 100,000 inhibits expression ofpro-apoptotic proteins such as Bid or Bax, which play an important rolein apoptosis of liver cell by D-GalN/LPS, was accomplished as shown inthe below.

700 mg/kg of D-GalN and 10 mg/kg of LPS were intraperitoneallyadministered to the mice. The fraction or physiological saline solutionwas intraperitoneally administered to the mice at 12 hours and 1 hourbefore administration of D-GalN/LPS. The said fraction was administeredto the mice in amount of 10 mg/kg, 30 mg/kg or 100 mg/kg. Liver cellisolated from the mice to which the said fraction or physiologicalsaline solution was administered was added to lysis buffer solution(prepared by mixing 50 mM Tris-HCl, 1% Nonidet P-40, 1 mM EDTA, 1 mMphenylmethyl sulfonyl fluroride, 1 g/ml leupeptin with 150 mM NaCl, pH7.5), then homogenized and centrifuged at 15,000 ×g for 10 mm to preparecrude protein. Total concentration of protein was measured by usingBradford method. 50 □ of protein was loaded at 12%˜15% sodium dodecylsulfate polyacrylamide gel. After electrophoration the said gel wastransferred to PVDF membrane (Millipore, Bedford, Mass. USA). Themembrane was incubated for 3 hours in the solution prepared by mixingTris-buffered saline solution and 0.1% Tween 20(Sigma corp.) and adding5% skim milk. Then, primary antibody such as rabbit polyclonal anti-Baxantibody (Santa Cruz Biochemicals, Santa Cruz, Calif. USA) or anti-Bidantibody (Santa Cruz Biochemicals, Santa Cruz, Calif. USA) was incubatedin the said membrane. Thereafter, the membrane was washed with bufferedsaline for 15 mm and three times. Thereafter, secondary antibody wasincubated in the membrane for 1 hour. The membrane was washed.Expression of protein was measured with Amersham ECL system (AmershamPharmacia Biotec, Buckinghamshire, UK).

FIG. 9 shows that the fraction containing polysaccharide with amolecular weight larger than 100,000 inhibited expression of Bax,pro-apoptotic protein concentration dependently. Also, the expression ofBid, pro-apoptotic protein in the fraction-treated group was similar tothat of Bid in normal group concentration dependently. However, in caseof the mice to which physiological saline solution was administered,expression of Bid was decreased relative to the result of normal group.Therefore, the fraction containing polysaccharide with a molecularweight larger than 100,000 inhibits activation or expression ofpro-apoptotic protein induced by liver-injury in liver cell.Consequently, the fraction has an efficacy on the liver protection.

EXPERIMENTAL EXAMPLE 8

Liver Protection in Oral Administration of the Extract of Acanthopanaxkoreanum to the Mice

The experiment confirming whether the 80%-ethanol insoluble part amongthe water extract has an efficiency for liver protection for oraladministration was accomplished as shown in the below.

The said 80%-ethanol insoluble part was dissolved in water. The solutionwas orally fed the mice in an amount of 100 mg/kg or 300 mg/kg per day.D-GalN/LPS was administered to the mice at one week after administrationof the said part. The lethality rate was measured for 24 hours. As forcontrol group to which the said part was not administered, the lethalityrate was measured for 24 hours.

As shown in FIG. 10 and 11, the control group was diseased at 6 hoursafter administration of D-GalN/LPS. All the mice of the control groupwere diseased at 12 hours after administration of D-GalN/LPS. However,three of six mice to which 300 mg/kg of the said ethanol insoluble partwas administered survived at 24 hours after administration ofD-GalN/LPS.

The said 80%-ethanol insoluble part inhibits the formation of TNF-α andexpression of various pro-apoptotic proteins. Therefore, the saidethanol insoluble part has an efficacy for the liver protection.

EXPERIMENTAL EXAMPLE 9

Acute Toxicity in Oral Administration of the Extract of Acanthopanaxkoreanum to the Mice

The experiment confirming whether the extract of the present inventionhas acute toxicity or not was accomplished as shown in the below.

As for the experimental group comprising of five or six mice (B57BL/6)having body weights of 20 g, the experiment confirming whether theextract of the present invention has acute toxicity or not wasaccomplished. The 80%-ethanol insoluble part among the water extract ofAcanthopanax koreanum prepared in example 2 was orally administered tothe experimental group in an amount of 2000 mg/kg, thereaftertoxification of the experimental group was observed for 7 days.Particularly, death, clinic symptoms, change of body weight of the micewere observed after administration of the said part. Also, hematologicaltest and biochemical test of blood were accomplished. The mice wereautopsied to test abnormality of abdominal cavity or pleural cavity. Inthe group to which the said part was administered, abnormality of changeof body weight, clinic symptoms, result of hematological test,biochemical test of blood, and autopsy was not observed. Also, the micewere not diseased.

The said ethanol insoluble part causes no toxic effect in all of themice. LD50 for oral administration is 2000 mg/kg. Therefore, the saidethanol insoluble part has high stability in the mice body. Also, thesaid ethanol insoluble part was safely administered to the body toprotect liver.

INDUSTRIAL APPLICABILITY

The said extract of the present invention has efficacy illustrated inthe below.

First, the present invention provides 1) the water extract ofAcanthopanax koreanum obtained by treating the root or stem ofAcanthopanax koreanum with water, 2) the ethanol insoluble part amongthe said water extract, obtained by treating the said water extract withethanol, 3) the fraction containing polysaccharide with a molecularweight larger than range of 12,000˜14,000, among the said ethanolinsoluble part, or 4) the fraction containing polysaccharide with amolecular weight larger than 100,000, among the said ethanol insolublepart.

Second, the extract of the present invention reduces the serum level ofAST, ALT and TNF-α in liver-injury mice model induced by D-GalN/LPS tobe similar to ones of normal group also, the extract of the presentinvention inhibits expression of pro-apoptotic protein of liver cellactivated by TNF-α Third, the extract of the present invention inhibitsthat DNA in liver cell is cleaved to the small fragments.

Forth, the extract of the present invention made high survival rate morethan 90%, maintained in the experiment for measurement of lethality rateprogressed for 24 hours. Also, the extract of the present invention hasno toxic effects in histological test. Therefore, the extract of thepresent invention can be used as therapeutic agent or preventor of thehepatitis, or a liver protective drug.

1. An ethanol insoluble part derived from a water extract of the root orstem of Acanthopanax koreanum for treatment of hepatitis or protectionof liver by inhibiting apoptosis of a liver cell, wherein the ethanolinsoluble part is comprised of polysaccharide with a molecular weightlarger than the range of 12,000˜14,000, and, wherein said ethanolinsoluble part is prepared by a process comprising the steps of: 1)obtaining said water extract of the root or stem of Acanthopanaxkoreanum prepared by extracting Acanthopanax koreanum with water; 2)adding ethanol to the water solution to produce the aqueous ethanolsolution, wherein the final concentration of ethanol in the aqueousethanol solution is between 50% and 90%; 3) centrifuging the aqueousethanol solution to precipitate; and, 4) filtering the ethanol insolublepart precipitated in the aqueous ethanol solution to obtain the ethanolinsoluble part.
 2. The ethanol insoluble part of claim 1, wherein saidethanol insoluble part is comprised of polysaccharide with a molecularweight larger than 100,000.
 3. The ethanol insoluble part of claim 1,wherein the final concentration of ethanol in step 3) is 80%.
 4. Apharmaceutical composition for the treatment of hepatitis containing theethanol insoluble part of any of claims 1, 2 and
 3. 5. A pharmaceuticalcomposition for protection of liver by inhibiting apoptosis containingthe ethanol insoluble part of any of claims 1, 2 and
 3. 6. A TNF-αinhibitor containing the ethanol insoluble part of any of claims 1, 2and
 4. 7. A Health supplementary food containing the ethanol insolublepart of any of claims 1, 2 and 4.